Nucleic acid probe-based diagnostic assays for prokaryotic and eukaryotic organisms

Abstract

Use of the ssrA gene or tmRNA, an RNA transcript of the ssrA gene, or fragments thereof as target regions in a nucleic acid probe assay for the detection and identification of prokaryotic and/or eukaryotic organisms is described. Nucleotide sequence alignment of tmRNA sequences from various organisms can be used to identify regions of homology and non-homology within the sequences which in turn can be used to design both genus specific and species specific oligonucleotide probes. These newly identified regions of homology and non-homology provide the basis of identifying and detecting organisms at the molecular level. Oligonucleotide probes identified in this way can be used to detect tmRNA in samples thereby giving an indication of the viability of non-viral organisms present in various sample types.

The invention claimed is: 
     
       1. A method of detecting and/or identifying Mycobacteria tuberculosis in a sample in vitro, which comprises, determining the presence of a nucleotide sequence encoding an ssrA gene from  Mycobacteria tuberculosis , wherein said isolated nucleic acid is selected from the group consisting of SEQ ID NOS: 67, 73, 184, 200, 219, 224, 225, or a nucleic acid encoding a tmRNA from  Mycobacteria tuberculosis , wherein said isolated nucleic acid is selected from the group consisting of SEQ ID NO: 68, 74, 185, 201, or a fragment thereof consisting of at least ten nucleotides. 
     
     
       2. The method according to  claim 1 , wherein a fragment of at least ten nucleotides of the ssrA gene or a fragment of at least ten nucleotides of the tmRNA, which corresponds to a region of high homology from the 5′ end or a region of high homology from the 3′ end of the DNA molecule is used as a universal target region. 
     
     
       3. The method according to  claim 1 , wherein a fragment at least ten nucleotides of the ssrA gene or a fragment of at least ten nucleotides of the tmRNA, which corresponds to a region of low homology is used as a target region in a nucleic acid probe assay to distinguish between species. 
     
     
       4. The method according to  claim 1 , wherein a fragment of at least ten nucleotides of the ssrA gene or a fragment of at least ten nucleotides of the tmRNA, which corresponds to a region of low homology is used as a target region for the generation of a genus specific probe. 
     
     
       5. The method according to  claim 1 , which comprises an amplification procedure wherein said ssrA gene fragment or said tmRNA fragment is used as the basis of a primer. 
     
     
       6. The method according to  claim 5 , further comprising obtaining a product of the amplification procedure and performing a nucleic acid probe assaying, wherein said product is used as a target region in the nucleic acid probe assay. 
     
     
       7. The method according to  claim 1 , which comprises a nucleic acid hybridization assay in which a cDNA transcript of the tmRNA is used as a probe. 
     
     
       8. The method according to  claim 1 , which comprises using a fragment of at least ten nucleotides of the ssrA gene or tmRNA as a target region and distinguishing living and dead organisms or broad scale detecting and/or identifying organisms using a multi-probe format. 
     
     
       9. The according to  claim 8  wherein an ssrA gene probe comprising at least ten nucleotides of the ssrA gene or a tmRNA transcript probe comprising at least ten nucleotides of the tmRNA is linked to a microarray gene chip system for the broad scale high throughput detection and identification of  Mycobacteria tuberculosis . 
     
     
       10. The method according to  claim 1 , which comprise detecting and/or identifying  Mycobacteria tuberculosis  in a sample of matter. 
     
     
       11. The method according to  claim 1 , further comprising obtaining a DNA profile of  Mycobacteria tuberculosis  to thereby distinguish between strains of Mycobacteria tuberculosis. 
     
     
       12. A method of monitoring the efficacy of drug therapies against Mycobacteria tuberculosis, which administering a drug to a patient in need thereof and detecting and/or identifying  Mycobacteria tuberculosis  with the method according to  claim 1  prior to and subsequent to the administration of the drug. 
     
     
       13. The method according to  claim 1  wherein the  Mycobacteria tuberculosis  is quantified.